Fluorescent Polystyrene Beads Research Articles

"Satellite cells" and nerve terminals in the crayfish opener muscle visualized with fluorescent dyes.

Nerve terminals and associated cells on the muscle's surface were visualized in the crayfish opener muscle with several fluorescent dyes in conjunction with confocal microscopy and conventional fluorescence microscopy. The nerve terminals of the excitatory and inhibitory axons were best seen with 4-diethylaminostyryl-N-methylpyridinium iodide (4-Di-2-Asp). This dye is selectively accumulated in mitochondria, which are numerous both in the axons and in synapse-bearing terminal varicosities. Muscle nuclei were also clearly visualized, because they excluded 4-Di-2-Asp but were stained by acridine orange (AO). A positive attraction between muscle nuclei and nerve terminals was evident by visual inspection and was confirmed by spatial statistics. Additional flat cells on the muscle's surface appeared as bright rings with elongated processes that were often close to or overlapped nearby nerve terminals. The structure of these cells was established by electron microscopy after labeling them with fluorescent polystyrene beads, which could be found over structures on the muscle surface in sections of embedded specimens. The flat surface cells were distinct from peripheral glial cells closely associated with axons and nerve terminals. Nevertheless, spatial statistics showed that the surface cells were grouped near nerve terminals. They occupied a small fraction of the muscle cell's surface. Their functional role has not been determined in crustacean muscles.

Paracrine downregulation of Fc gamma RIII in human monocyte-derived macrophages induced by phagocytosis of nonopsonized particles

Monocytes and macrophages can take up nonopsonized particles through a direct phagocytic process and IgG-coated particles through combined mechanisms of nonspecific phagocytosis and internalization mediated by specific Fc receptors for IgG (Fc gamma R) on the plasma membrane. In this study, we report the effect of phagocytosis of nonopsonized latex beads on the levels of expression of Fc gamma receptors in human monocyte-derived macrophages (MDM) as measured by flow cytometry (fluorescence-activated cell sorter [FACS]). Macrophages were exposed to green fluorescent 1-micron polystyrene beads before labeling for Fc gamma RI, Fc gamma R-II, and Fc gamma R-III, respectively, with 32.2, 2E1, and 3G8 monoclonal antibodies (MoAbs) and a phycoerythrin (PE)- conjugated secondary Ab to permit dual-channel analysis of fluorescence intensity by FACS. Macrophages that had phagocytosed at least one bead showed reduced levels of surface Fc gamma R-III but not Fc gamma R-I or Fc gamma R-II when compared with cells that had never been exposed to beads. Moreover, cells that were not in direct contact with beads, but that shared medium with cells that had phagocytosed beads also had reduced levels of Fc gamma R-III but not Fc gamma R-I or Fc gamma R-II, suggesting a cytokine-mediated mechanism of Fc gamma R-III downregulation. Phagocytosis of 1-micron beads alone stimulated macrophages to release tumor necrosis factor-alpha (TNF-alpha). The medium from macrophages phagocytosing beads could stimulate other macrophages not in direct contact with beads to release TNF-alpha as well, but such paracrine-triggered release could be reduced by more than 50% if the medium from the phagocytosing cells was first treated with a neutralizing anti-TNF-alpha antibody. Moreover, the paracrine downregulation of Fc gamma R-III described above could also be blocked if the neutralizing anti-TNF-alpha antibody was added to the medium of phagocytosing cells. Treatment of macrophages with recombinant human TNF-alpha in the absence of beads induced decreased levels of Fc gamma R-III but not of Fc gamma R-II. These results show that paracrine downregulation of Fc gamma R-III is mediated by a TNF-alpha-dependent mechanism.

Controlled release using ionotropic polyphosphazene hydrogels

Poly(bis(carboxylatophenoxy)phosphazene) (PCPP) forms hydrogels when treated in aqueous media with salts of divalent cations, such as calcium chloride. This system has potential as a material for the controlled release of drugs. To study the ability of the PCPP hydrogel matrix to release macromolecular substrates, 24-nm fluorescent polystyrene beads or proteins with varying molecular weights were encapsulated in polyphosphazene microspheres. The influence of polyphosphazene concentration and ionic crosslinker content on the outward diffusion of substrates and on microsphere morphology was investigated. In order to create a poly- L-lysine (PLL) outer membrane coating around the PCPP microspheres, the spheres were treated with PLL solution (single coating) or sequentially with PLL and PCPP solutions (double coating). The formation of these membranes (stable poly electrolyte complexes) strongly influences the permeability of the polyphosphazene microspheres and contributes to their stability in physiological saline solution. This enables liquefaction and swelling of the internal core of the microcapsules in saline solution via ion-exchange reactions with monovalent salts and chelating agents. Increasing the PLL molecular weight in the coating process results in a significant enhancement of the substrate release rate. It is possible that the mechanism of this process includes partial coating rupture without loss of bead coherence. The effect of microsphere coating on release profiles can be controlled by varying the molecular weights, the concentration of PLL and reaction times between PCPP microspheres and PLL.

Human macrophage (M phi) polymorphism demonstrated by differential phagocytosis of fluorescent polystyrene beads.

Human macrophage (M phi) polymorphism demonstrated by differential phagocytosis of fluorescent polystyrene beads.

Uptake and tissue distribution of abiotic particles from the alimentary tract of the American oyster: A simulation of intracellular parasitism

Windows were cut in the left valves of American oysters, Crassostrea virginica, to access the mouth. A suspension of 2.16-μm-diameter fluorescent polystyrene beads were introduced into their stomachs by intubation and the windows were sealed. Stomach contents were sampled and vibratome cross sections were made at various time intervals postintubation. The locations of the beads in the tissues were mapped using an image analysis system. Hemocytes containing phagocytized beads were found in the lumen of the stomach shortly after intubation. Hemocytes with internalized beads were clustered in the tissues around the gut, in the mantle cavity, and in the palps at 2 hr postintubation. At 2 and 5 days after intubation the intracellular beads were randomly scattered in the tissues and not necessarily associated with the digestive tract. The number of beads present in the tissues diminished with time but some remained in the tissues 10 days after intubation. The polystyrene beads simulate to some extent parasites, which are phagocytized but are not destroyed by the hemocytes. These data show that indigestible particles carried in the phagosomes of hemocytes can enter the tissues from the stomach. The phagocytized particles are gradually eliminated from the tissues, but a number remain residual in the tissues within hemocytes for more than 10 days.

Occurrence of bacterivory in Cryptomonas, a common freshwater phytoplankter

Bacterivory was detected by incorporation of 0.57 μm diameter, fluorescent polystyrene beads and fluorescently labeled bacteria (FLB) in two cultured species of Cryptomonas (C. ovata and C. erosa), and a population of Cryptomonas sp in a humic, mesotrophic lake. Rates of ingestion and clearance were very low, and similar for the cultures and the in situ population. The in situ population incorporated 0.7-1.7 bacteria cell-1 h-1, thereby ingesting 0.3%-2.0% of the total bacterial numbers present in the water per day, and receiving less than 2% of its carbon content per day through bacterivory. Thus, bacterivory by Cryptomonas was quantitatively important neither as a sink for bacterial biomass, nor as a carbon source for the algal cells. Possibly, it served in the uptake of essential nutrients.

Poly(I):poly(C)-enhanced alveolar and peritoneal macrophage phagocytosis: quantification by a new method utilizing fluorescent beads.

Phagocytosis is an important immune function to quantify. This immune response may be modulated by exposure to biological response modifiers or by exposure to pollutants. A new technique for quantifying nonspecific phagocytosis of alveolar and peritoneal macrophages in the same animal has been developed that utilizes fluorescent polystyrene beads. When incorporated into inhalation studies, this technique can be used to determine whether the toxic effect of an inhaled pollutant is local (effect on alveolar macrophages), systemic (effect on peritoneal macrophages), or both local and systemic. This method results in a determination of both the level of phagocytosis (the percentage of phagocytic macrophages) and the macrophage specific activity (the number of beads phagocytized per macrophage). This method also allows a determination of adherence by quantifying the number of particles in contact with, but not phagocytized by, the macrophage. Macrophage preparations were incubated with fluorescent beads for 2 hr and cyto-centrifuged onto a glass slide. Fluorescent beads present on the slide or cell-associated but not ingested by phagocytosis were removed by immersing the slide containing the macrophage preparation in methylene chloride for 15-30 sec. Fluorescent beads ingested by phagocytosis were then easily quantified with a fluorescence microscope. This technique was used to assess the baseline levels of phagocytosis for rat alveolar and peritoneal macrophages from the same animal and the kinetics and level of enhanced phagocytosis for alveolar and peritoneal macrophages after injection with the interferon inducer polyinosinate-polycytidylate (poly(I):poly(C)). The kinetics of enhanced alveolar and peritoneal macrophage phagocytosis by poly(I):poly(C) were similar; however, stimulated phagocytic levels of peritoneal macrophages never reached the phagocytic activity observed for the resident, highly phagocytic alveolar macrophages. This elevated phagocytic activity is most likely due to interferon stimulated by particulate matter in the large volume of air processed by the lungs and is important for host defense against a number of different inhaled microorganisms.

The application of flow cytophotometry in measurements of cell adhesion.

A common approach to the study of cell substrate interactions is the measurement of the attachment of cells to different substrates or to cultured cell layers. The evaluation of attachment is made either by scintillation counting of previously labelled adhering cells, or by light microscopy using the criterion of cell shape, sometimes refined by automatic image analysis. These methods have many drawbacks. This paper suggests the use of fluorescence-activated flow cytophotometry, (FC) which yields direct counts of the non-adhering cells. These "free" cells are removed after completion of the adhesion experiment from the microtitre plate wells. An internal standard, in the form of fluorescent polystyrene beads is added, allowing evaluation of the percentage of cells adhering to the well walls. Flow cytophotometry then produces data based on the analysis of large populations of cells. Unequivocal discrimination is obtained between the counted cells and counted fluorescent beads eliminating counting errors. The results can be processed on line by computer. A suspension of mouse splenocytes was used for the evaluation of the overall error of the method arising from inaccuracies in pipetting, interference of glutaraldehyde with ethidium bromide (EB) staining and instrumental error. Each adhesion experiment was terminated by staining and post-fixation and it was established that this introduces no change in cell counting, in comparison with the original unfixed cells. Prefixation, however, quenches the EB staining and would interfere with the counting procedure. The overall standard error of the technique was found to be 5%-10%.(ABSTRACT TRUNCATED AT 250 WORDS)

Microinjected fluorescent polystyrene beads exhibit saltatory motion in tissue culture cells.

Microinjected 0.26-micron fluorescent, carboxylated microspheres were found to display classical saltatory motion in tissue culture cells. The movement of a given particle was characterized by a discontinuous velocity distribution and was unaffected by the activity of adjacent particles. The microspheres were translocated at velocities of up to 4.7 micron/s and sometimes exhibited path lengths greater than 20 micron for a single saltation . The number of beads injected into a cell could range from a few to over 500 with no effect on the cell's ability to transport them. Neither covalent cross-linking nor preincubation of the polystyrene beads with various proteins inhibited the saltatory motion of the injected particles. The motion of the injected beads in cultured cells was reversibly inhibited by the microtubule poison nocodazole, under conditions in which actin-rich, nitrobenzoxadiazol - phallacidin -staining structures remain intact. Whole-cell high voltage electron microscopy of microinjected cells that were known to be moving the fluorescent microspheres revealed that the beads were embedded in the cytoplasmic matrix and did not appear to be membrane bound. The enhanced detectability of the fluorescent particles over endogenous organelles and the ability to modify the surfaces of the beads before injection may enable more detailed studies on the mechanism of saltatory particle motion.

Isolation of human fibroblast heterokaryons with two-colour flow sorting (FACS II)

Red and green fluorescent polystyrene beads were used to label different populations of cultured human skin fibroblasts. After co-cultivation for 24 h no exchange of label could be observed. Twenty-four hours after fusion the population of red-green heterofluorescent cells was sorted out with a FACS II cell sorter. Microscopical examination of the heterofluorescent population 20 h after sorting indicated, that with Sendai-induced fusion, 50–60% of these viable cells contained more than one nucleus. After polyethylene glycol (PEG)-induced fusion, between 70 and 85% bi- or multinucleated cells were present in the sorted and cultured fraction. Autoradiography using [ 3H]thymidine indicated that the sorted heterofluorescent bikaryons were in fact heterokaryons.